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Department of Pharmacology, New York Medical College, Valhalla, New York 10595
Submitted 17 March 2003 ; accepted in final form 19 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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-nitro-L-arginine methyl ester. This
indicated that the effect of TNF was not mediated by a nitric oxide-dependent
pathway. Also, inhibition of PKA did not affect the stimulatory effect of TNF.
In contrast, inhibition of protein tyrosine kinase not only increased activity
of the 70-pS K+ channel but also abolished the effect of TNF.
Moreover, inhibition of protein tyrosine phosphatase (PTP) blocked the
stimulatory effect of TNF on the 70-pS K+ channel. The notion that
the TNF effect results from stimulation of PTP activity is supported by PTP
activity assay in which treatment of mTAL cells with TNF significantly
increased the activity of PTP. We conclude that TNF stimulates the 70-pS
K+ channel via stimulation of PTP in the mTAL. ROMK channel; protein tyrosine kinase; protein tyrosine phosphatase; NG-nitro-L-arginine methyl ester
The apical K+ channels play an important role in K+ recycling, which is essential for maintaining the normal function of Na-K-Cl cotransporter in the TAL (11). There are at least three types of K+ channels: Ca2+ activated, large conductance, and 30 and 70 pS (3, 13, 29). It is generally accepted that the 70- and 30-pS K+ channels are major K+ channels responsible for K+ recycling (30). In addition, it is possible that ROMK is an important component of both 70- and 30-pS K+ channels because neither K+ channel can be found in ROMK knockout mice (18). We showed that apical K channels are regulated by a variety of signaling pathways, including nitric oxide (NO), protein kinase C, and protein tyrosine phospatase (PTP) (12, 19, 32). TNF has been shown to activate these same pathways (1, 5, 16, 20, 35). Thus the main goal of the present study is to investigate the acute effect of TNF on apical K+ channels in the mTAL and delineate the mechanism by which TNF regulates the apical K+ channels.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Patch-clamp technique. Electrodes were pulled with a Narishige
model PP83 vertical pipette puller and had resistances of 4-6 M
when
filled with 140 mM NaCl. The channel current recorded by an Axon200A
patch-clamp amplifier was low-pass filtered at 1 kHz using an eight-pole
Bessel filter (902LPF, Frequency Devices, Haverhill, MA). The current was
digitized by an Axon interface (Digitada1200), collected by an IBM-compatible
Pentium computer (Gateway 2000) at a rate of 4 kHz, and analyzed using the
pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channel
activity was defined as NPo, a product of channel open
probability (Po) and channel number (N). The
NPo was calculated from data samples of 60-s duration in
the steady state as follows
 | (1) |
Cell cultures and measurement of protein tyrosine kinase and PTP
activity. Cultured mTAL cells were used to measure the activity of
protein tyrosine kinase (PTK) and PTP before and after treatment with TNF. The
method for isolation and primary culture of mTAL cells has been previously
described (28). The TAL cells
were incubated in the presence or absence of TNF (10 nM) for 10 and 15 min. We
followed the method described previously to measure PTK
(31). Cells were lysed in 100
µl of RIPA buffer [150 mM NaCl, 50 mM Tris·HCl (pH 7.4), 50 mM
-glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM EDTA, 5
mM EGTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate,
0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5
µg/ml leupeptin, and 2 µg/ml pepstatin]. For activity assay of PTK, 10
µl of the sample were incubated in a total volume of 30 µl containing
200 µM[32P]ATP (1 cpm/fmol), 12 mM magnesium acetate, 2 mM
MnCl2, 0.3 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.5 mM
ammonium molyb-date, and 2 mM R-R-Src peptide
(Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly). Reactions were quenched by
adding 200 ml of 75 mM phosphoric acid, and 15 µl of cocktail were spotted
on phosphocellulose paper. After several washes, the amount of 32P
incorporated into R-R-Src peptide was assessed using a liquid scintillation
counter.
We used 32P-labeled myelin basic protein described originally by Tonks et al. (27) as a substrate for the measurement of PTP activity. The release rate of 32P from the basic protein is used as an index of PTP activity, and the value obtained in the presence of vanadate is used as background. The activity of PTP was measured with a BioLabs PTP assay kit following the instruction provided by the vender (Bio-Rad, Hercules, CA). Briefly, cultured mTAL cells were incubated in the presence or absence of TNF (10 nM) for 10 min. After treatment, cells were lysed in 100-µl buffer solution containing 50 mM NaCl, 50 mM Tris·HCl (pH 7.4), 2.5 mM EDTA, 2.0 mM dithiothreitol, 0.01% Brij 35, 1.0 µg/ml aprotinin, 1.0 µg/ml leupeptin, 1.0 µg/ml pepstatin, and 2 mM phenylmethyl sylfonyl fluoride. The mixture (10 µl) was added to a tube containing 30-µl assay buffer composed of 1 mM NaCl, 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 1 mg/ml BSA at 30°C for an additional 5 min. Ten microliters of the 32P-labeled myelin basic protein were added to the sample followed by incubation for 10 min at 30°C. Reactions were terminated by adding 200 µl of ice-cold 20% trichloride acetate into the tube. The sample was centrifuged for 5 min at 12,000 g, the resultant supernatant was carefully taken, and the amount of 32P released from the peptide was measured using a liquid scintillation counter.
Solution and statistics. The pipette solution was composed of (in mM) 140 KCl, 1.8 Mg2Cl, and 5 HEPES (pH = 7.4). NG-nitro-L-arginine methyl ester (L-NAME) and phenylarsine oxide were purchased from Sigma (St. Louis, MO), whereas H8 and herbimycin A were obtained from Biomol (Plymouth Meeting, PA). Herbimycin A was dissolved in DMSO and the final concentration of DMSO was <0.1% that had no effect on channel activity. TNF was obtained from PeproTech (Princeton, NJ) and diluted in sterile water at a concentration of 29 µM as a stock solution. The data are means ± SE. We used paired and unpaired Student's t-tests to determine the statistical significance. If the P value was <0.05, the difference was considered to be significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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1 nM
(Fig. 2). In addition to the
70-pS K+ channel, a ROMK-like 30-pS K+ channel was also
expressed in the mTAL (3,
18,
29). We examined the effect of
10 nM TNF on the ROMK-like 30-pS K+ channel. To determine the
effect of TNF on the 30-pS K+ channel, patches containing the 30-pS
K+ channels with a relatively low Po were
selected. Figure 3 is a
recording made in a cell-attached patch demonstrating that TNF did not affect
the activity of the 30-pS K+ channel (n = 3) because
NPo = 0.51 ± 0.05 (TNF) was not different from 0.50
± 0.05 (control).
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As the dose-response curve of TNF was established, 5 nM TNF was used in experiments designed to study the mechanism by which TNF stimulates the 70-pS K+ channel. Several factors that have been shown to stimulate the 70-pS K+ channel include protein kinase A (PKA), the NO-cGMP-dependent pathway, and PTP (12, 19, 29). Therefore, we examined the effect of TNF on the 70-pS K+ channel in the presence of PKA inhibitors (Fig. 4). Inhibition of PKA did not significantly affect channel activity. Moreover, inhibition of PKA did not alter the TNF effect because application of TNF further increased channel activity from 0.3 ± 0.03 to 1.10 ± 0.1 (n = 5) in the presence of H8. This suggested that the stimulatory effect of TNF does not result from stimulation of PKA.
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Stimulation of TNF receptors has been shown to increase NO production (5, 16). Because the NO-dependent cGMP pathway can stimulate activity of the 70-pS K+ channel, we investigated the role of NO in mediating the effect of TNF on the 70-pS K+ channel in the presence of L-NAME, an inhibitor of NO synthase (NOS). Inhibition of NOS decreased the NPo of the 70-pS K+ channel to 0.13 ± 0.02 (n = 5). This is consistent with the previous observation that L-NAME inhibited activity of the 70-pS K+ channel (19). However, L-NAME did not block the effect of TNF because it increased NPo from 0.13 ± 0.02 to 0.9 ± 0.1 (n = 5) in the presence of 0.5 mM L-NAME (Fig. 5).
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Several studies demonstrated that stimulation of the TNF receptor increases PTP (1, 26). We previously observed that the 70-pS K+ channel is regulated by PTK and PTP (12). If the effect of TNF was the result of stimulation of PTP, which leads to enhancement of tyrosine dephosphorylation, inhibition of PTK should mimic the effect of TNF such that the effect of TNF on the 70-pS K+ channel activity should be absent in the presence of a PTK inhibitor such as herbimycin A. This possibility has been tested by experiments in which the effect of TNF was examined in the tubule treated with herbimycin A. Figure 6 is a recording showing the effect of TNF on channel activity in the presence of herbimycin A. We confirmed the previous finding that inhibition of PTK significantly stimulates the 70-pS K+ channel (12) and increases NPo from 0.24 ± 0.02 to 1.22 ± 0.1 (n = 5). Furthermore, in the presence of herbimycin A, the stimulatory effect of TNF on the 70-pS K+ channel is absent because TNF did not significantly increase channel activity (NPo = 1.25 ± 0.1). This indicates that the effects of TNF and herbimycin A are not additive.
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Although the observation that the effects of TNF and herbimycin A are not additive supports the notion that the effect of TNF on the 70-pS K+ channel is mediated by stimulation of PTP activity, it is possible that TNF cannot further increase channel activity because it is already at peak following inhibition of PTK. Therefore, we examined whether inhibition of PTP can reverse the stimulatory effect of TNF. Because addition of phenylarsine oxide (PAO), an inhibitor of PTP, decreased channel activity, we selected the patches with a high NPo to study the effect of TNF in the presence of PAO. Inhibition of PTP not only decreased NPo from 0.44 ± 0.04 to 0.21 ± 0.01 (n = 5) but also abolished the effect of TNF on channel activity because NPo did not significantly alter and was 0.18 ± 0.01 (Fig. 7). Moreover, we examined the effect of PAO on channel activity in the mTAL, which was challenged with TNF. Application of TNF increased channel activity from 0.24 ± 0.02 to 0.92 ± 0.1 (Fig. 8). However, in the presence of TNF, addition of PAO reduced channel activity from 0.92 ± 0.1 to 0.1 ± 0.01 (n = 5) (Fig. 8). This further suggests that the effect of TNF on the 70-pS K+ channel is mediated by stimulation of PTP.
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To examine whether TNF treatment can stimulate the activity of PTP, we used cultured mTAL cells (28) to measure the activity of PTP in the presence or absence of 10 nM TNF for 10 min. Results summarized in Fig. 9 show that treatment of mTAL cells with 10 nM TNF increased the release of 32P from the substrate from 2,050 ± 100 pmol/mg protein (control) to 4,200 ± 300 pmol/mg protein (n = 4). The effect of TNF on PTP was specific because TNF did not alter the activity of PTK (control 35,777 ± 2,000 pmol/mg protein, TNF 35,250 ± 2,100 pmol/mg protein, n = 5) in mTAL cells.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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PTK and PTP have been shown to play important roles in the regulation of the 70-pS K+ channel: stimulation of PTK decreases, whereas an increase in PTP activity augments activity of the 70-pS K+ channel (12). The effect of PTK on the 70-pS K+ channel is possibly the result of the stimulation of tyrosine phosphorylation of the 70-pS K+ channel or its closely associated proteins because addition of exogenous c-Src PTK could inhibit the 70-pS K+ channel in inside-out patches (12). We also observed that TNF did not inhibit the ROMK-like 30-pS K+ channel in the TAL. This suggests that the 30-pS K+ channel is not directly regulated by PTP. The finding is consistent with previous findings that stimulation of PTK activity inhibits only the 70-pS K+ channel but not the 30-pS K+ channel (12). Therefore, the mechanism by which PTP or PTK regulates the apical 30-pS K+ channel is different from that of the 70-pS K+ channel. In addition, the observation that inhibition of PTP decreased the ROMK-like 35-pS K+ channel in the cortical collecting duct (CCD) (34) indicates further that ROMK-like small-conductance K+ channels in the mTAL and CCD are also differentially modulated by PTK and PTP. Because ROMK1 is in the CCD whereas ROMK2/3 is in the TAL (4), this strongly suggests that the mechanism by which PTK regulates ROMK1 or ROMK2/3 is different. The physiological significance of the finding that only the 70-pS but not the 30-pS K+ channel is regulated by TNF is not clear. It is speculated that the 30-pS K+ channel may be responsible for providing a basal level of apical K+ conductance, whereas the 70-pS K+ channel activity is subjected to modulation by a variety of factors such as TNF.
TNF is a cytokine produced by a variety of cells including the epithelial cells in the mTAL (21). TNF production increases in response to inflammation, injury, and infection. It has been reported that IL-1 and -2 increase TNF production (20). TNF plays an important role in inflammation, cell differentiation, and cell death (20). The role of TNF in renal pathophysiological mechanisms has been well established. For instance, TNF may contribute to the chronic tubule injury associated with hypercalcemia. Moreover, TNF can be produced by physiologically relevant stimulations. It has been shown that stimulation of the Ca2+-sensing receptor increases TNF generation (15, 28).
Stimulation of the Ca2+-sensing receptor is expected to decrease Na+ reabsorption in the mTAL (14). One mechanism by which stimulation of the Ca2+-sensing receptor inhibits Na+ transport is to block K+ recycling across the apical membrane in the mTAL. We previously demonstrated that raising extracellular Ca2+ inhibits the apical 70-pS K+ channel by increasing 20-HETE generation (33). Because K+ recycling is important, inhibition of K+ channels should lead to decreased Na+ absorption in the mTAL (11). However, this 20-HETE-dependent mechanism may be responsible for acute stimulation of the Ca2+-sensing receptor, whereas the TNF-cyclooxygenase (COX)-dependent pathway is involved in mediating the effect of sustained stimulation of the Ca2+-sensing receptor in the mTAL. It has been shown that a prolonged stimulation of the Ca2+-sensing receptor by incubation of mTAL cells in a media containing high Ca2+ for more than 3 h increases COX-2 expression and PGE2 generation. TNF has been shown to play a key role in inducing COX-2 following stimulation of the Ca2+-sensing receptor (28). Because PGE2 has been shown to inhibit the apical K+ channel (17) and other ion transporters in the mTAL (7), an increase in PGE2 production following stimulation of the Ca2+-sensing receptor should decrease Na+ transport in the mTAL. Therefore, COX-dependent metabolites of arachidonic acid are likely involved in mediating effects of a sustained stimulation of the Ca2+-sensing receptor. Although TNF stimulates the apical 70-pS K+ channel, it is unlikely that the net effect of prolonged stimulation of the Ca2+-sensing receptor would increase channel activity. This is due to the fact that increases in TNF production induced by raising extracellular Ca2+ may not be high enough to significantly stimulate the 70-pS K+ channel. It was calculated that the concentration of TNF in the mTAL incubated in a media containing 2 mM Ca2+ was below 10 pM (28). Because TNF at concentrations lower than 10 pM does not affect channel activity (Fig. 2), the net effect of long-lasting stimulation of the Ca2+-sensing receptor should be inhibiting the apical K+ channel.
The finding that TNF can be formed in the mTAL in which 20-25% of filtered Na+ load is reabsorbed strongly suggests that TNF may have an effect on membrane transport in the mTAL. This notion is supported by observations that incubation of isolated mTAL in TNF containing media for 24 h decreased ouabain-sensitive Rb+ uptake (10). TNF-induced decreases in ouabain-sensitive Rb+ uptake suggest that TNF inhibits the active Na+ transport in the mTAL. In the present study, we observed that TNF increases the 70-pS K+ channel activity. Because the 70-pS K+ channel contributes significantly to the apical K+ conductance and K+ recycling (30), it is expected that TNF should increase Na+ influx across the apical membrane. This apparent paradox may best be explained by considering that effects of TNF are time dependent and biphasic: the acute effect of TNF is to stimulate, whereas the chronic effect of TNF is to inhibit the active Na+ transport in the mTAL. Also, the acute effect of TNF is mediated by stimulation of PTP. This is a direct consequence of stimulation of TNF receptor-dependent signaling without the involvement of gene transcription. In contrast, the chronic effect of TNF on Rb+ uptake is the result of increasing COX-2 expression and activity because blocking COX-2 attenuates the inhibitory effect of TNF on Rb+ uptake (10, 28). Therefore, the effect of TNF on the transport in the mTAL depends not only on concentrations but also on temporal factors. A similar phenomenon that the response of cellular signaling to hormones depends on exposure time or pattern has been reported in mediating the effect of growth factor (2).
The role of TNF in the regulation of epithelial transport in the mTAL is not clear. Because TNF production is stimulated by bacterial products and cytokines, it is possible that TNF may be responsible for the abnormal renal Na+ handling during pathological conditions. In this regard, it has been reported that Na+ excretion is reduced, whereas TNF production increased in diabetic rats (9). Furthermore, TNF stimulated Na+ uptake in the distal tubule cells isolated from diabetic rats (9). Therefore, it is conceivable that TNF may also increase Na+ transport under certain circumstances in the mTAL by stimulation of K+ recycling. Moreover, it has been reported that TNF-induced cell apoptosis is related to activation of K+ channels, which leads to intracellular K+ loss (23, 24). Indeed, inhibition of K+ channels has been demonstrated to prevent cell death in the proximal tubule induced by hypoxia (25). However, further study is required to examine whether activation of the apical K+ channels is the early event of TNF-induced cell damage in the mTAL.
In conclusion, acute application of TNF stimulates the apical 70-pS K+ channel and the effect of TNF is most likely mediated by enhancing PTP activity in the mTAL.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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